Antiproliferative Activity of (−)‐Isopulegol‐based 1,3‐Oxazine, 1,3‐Thiazine and 2,4‐Diaminopyrimidine Derivatives

Abstract A series of novel heterocyclic structures, namely 1,3‐oxazines, 1,3‐thiazines and 2,4‐diaminopyrimidines, were designed and synthesised. The bioassay tests demonstrated that, among these analogues, 2,4‐diaminopyridine derivatives showed significant antiproliferative activity against different human cancer cell lines (A2780, SiHa, HeLa, MCF‐7 and MDA‐MB‐231). Pyrimidines substituted with N 2‐(p‐trifluoromethyl)aniline, in particular, displayed a potent inhibitory effect on the growth of cancer cells. Structure–activity relationships were also studied from the aspects of stereochemistry on the aminodiol moiety as well as exploring the effects of substituents on the pyrimidine scaffold.

H2 atmosphere (1 atm) at room temperature. After completion of the reaction (as monitored by TLC, 24 h), the mixture was filtered through a Celite pad, and the solution was evaporated to dryness. The crude product was recrystallised in Et2O, resulting in primary aminoalcohols 8cd as white crystals.

General procedure for the preparation of thioureas (15ad)
Aminodiols 2a, 6, 10 and 12 (0.53 mmol) and the appropriate phenylisothiocyanate (0.79 mmol) were dissolved in toluene (40 mL), and the mixture was stirred at room temperature for 1 h, except that in the case of 6 when a treatment at reflux temperature for 3 h was carried out. The resulting mixtures were then evaporated then the residue was purified by column chromatography on silica gel (eluted with CHCl3 : MeOH = 19:1). -2-((1R,2R,4R)-2-hydroxy-4-methylcyclohexyl)

General procedure for the preparation of 1,3thiazines (16ab)
A solution of thioureas 15b or 15d (0.31 mmol) in dry EtOH (1 mL) was added 22% HCl in EtOH (5 mL) and the mixture was stirred at room temperature for 4 h and then concentrated under vacuum. The residue was treated with 10% KOH in MeOH (20 mL) followed by evaporation, and the crude product was again dissolved in water (10 mL) and extracted with CHCl3 (3 × 20 mL). The combined organic layer was washed with saturated NaCl aqueous solution (15 mL), dried (Na2SO4) and concentrated under vacuum. The crude product was purified by column chromatography on silica gel with CHCl3:MeOH = 19:1. 3-thiazinan-5-yl) 3-thiazinan-5-yl)

General procedure for the synthesis of 1.3-oxazines (17ad)
To a solution of 15ad (0.31 mmol) in MeOH (4 mL), MeI (1.50 mmol) was added. After 3 h stirring at room temperature, the mixture was evaporated, followed by adding 2.5 M KOH in MeOH (20 mL) and subsequently stirred for 1 h before evaporation. The residue was dissolved in water (20 mL) and extracted with CHCl3 (3 × 20 mL). The organic phase was then dried with Na2SO4 and evaporated to dryness. The crude product was purified by column chromatography on silica gel (CHCl3:MeOH = 19:1).

Determination of antiproliferative effect
The growth-inhibitory effects of the presented heterocyclic compounds were determined by a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on a panel of human adherent cancer cell lines of gynecological origin containing HeLa and SiHa (cervical cancers), A2780 (ovarian cancer) and MDA-MB-231 (breast cancer) cells. [7] All cell lines were purchased from the European Collection of Cell Cultures (Salisbury, UK) except the SiHa, obtained from the American Tissue Culture Collection (Manassas, VA, USA). The cells were cultivated in minimal essential medium (MEM) supplemented with fetal bovine serum (10%), non-essential amino acids, and penicillin-streptomycin (1% each) at 37 °C in a humidified atmosphere containing 5% CO2. All media and supplements were obtained from Lonza Group Ltd. (Basel, Switzerland). Cancer cells were plated into 96-well plates at the density of 5000 cells/well. After overnight incubation, the test compound was added in two concentrations (10 µM and 30 µM) and incubated for 72 h under cell-culturing conditions. Then, MTT solution (5 mg/mL, 20 μL) was added to each well and incubated for four h. Finally, the medium was removed, and the precipitated formazan was dissolved in DMSO during 60 min of shaking at 37 °C. The absorbance was measured at 545 nm using a microplate reader (SpectoStarNano, BMG Labtech, Ortenberg, Germany). Two independent experiments were carried out with five wells for each condition. Cisplatin (Ebewe GmbH, Unterach, Austria) was used as a positive control. Calculations were performed utilising the GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA). During our antiproliferative work we intended to use a practical investigational approach, therefore, we focused on the present treatment guidelines of gynecological cancers to choose a long time available, well-characterised positive control molecule which is recommended for the therapy of all cancer types included in our experiment (namely, breast, cervical and ovarian cancer). According to the online available guidelines of the European Society of Medical Oncology (ESMO) platinum compounds, like cisplatin, are effective in the clinical treatment of different gynecological cancers.

Investigation of antiproliferative activity of the ()-isopulegol-based heterocyclic compounds
Platinum-based therapy is still the first-line treatment option in local cervical cancer and it can be beneficial in combination with paclitaxel or topotecan in the therapy of advanced/metastatic cervical cancer. [8] Moreover, patients suffering from either primary (early stage or epithelial form) or recurrent ovarian cancer can be treated with platinum derivatives, in most cases as part of combination chemotherapy. [9] Among numerous different types of breast cancer according to their pathophysiological or developmental origin, for most triple negative breast cancer (defined by the absence of expression of estrogen and progesterone receptors and of overexpression of HER2 or amplification of HER2neu) chemotherapy included platinum derivatives remains the standard treatment. [10] MDA-MB-231 cell line used in our work is an experimentally applicable triple negative breast cancer cell line.

Docking study
According to the validation study, the RMSD values showed that the crystal structure 4DEE (ADP bound to Aurora A active site) is valid and could be confidingly used for docking study, where the RMSD values of all ADP generated conformations were less than 2 A° (Table 2).

Determination of Molecular properties of 20b
Based upon the truth that oral bioavailability is highly affected by the molecular properties of the drug molecule, we have calculated the molecular properties of compound 20b by using Accelrys Discovery Studio 2.5 software. The Lipinski's rules of five shows that the absorption of a ligand is higher when: molecular weight less than 500 D, calculated log P value less than

A B
Compound 20b C 5, no more than 5 hydrogen bond donor (HBD) groups, no more than 10 hydrogen bond acceptor (HBA) groups, and no more than 10 rotatable bonds.
As shown in table (3), it is obvious that compound 20b in accordance with Lipinski's rules of five, so it might have good oral bioavailability.

In-Silico ADMET analysis
ADMET refers to absorption, distribution, metabolism, excretion and toxicity properties for a molecule. They were predicted for compound 20b by using ADMET descriptors in Accelrys Discovery studio 2.5. There are six mathematical models are used to quantitatively predict properties of a set of compounds. These models contain: aqueous solubility (predict solubility in water at 25ºC, blood brain barrier (BBB) penetration, cytochrome P450 (CYP450) 2D6 inhibition, hepatotoxicity, human intestinal absorption (HIA) and plasma protein binding. [11] An ADMET model was also generated to predict the human intestinal absorption (HIA) and Blood Brain Barrier (BBB) penetration of tested compound. The model includes 95 and 99% confidence ellipses in the ADMET_PSA_2D and ADMET_ALogP98 plan as shown in figure (2).  As shown in Table 4, all these values are within the standard range shown in figure (2), which confirm that this compound has good absorption through human intestinal. Additionally, it exhibits low ability to penetrate blood brain barrier (BBB).